Semi-quantitative lateral-flow immunoassay for the detection of CSF leaks

ABSTRACT

Devices and methods are provided for the detection of CSF in a biological sample. In certain embodiments the device is a lateral flow device comprising: a porous substrate; a sample addition zone disposed on or in said porous substrate; a detection zone disposed on or in said porous substrate where said detection zone comprises at least a first test line (T 1 ) and a second test line (T 2 ) each test line comprising binding moieties that bind a complex formed between beta-trace protein (betaTP) and an indicator attached to a betaTP binding molecule; wherein said porous substrate defines a flow path through which a sample applied to the sample addition zone flows under capillary action away from said sample addition zone into said detection zone.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. 371 National Phase of PCT/US2018/024392,filed on Mar. 26, 2018, which claims benefit of and priority to U.S.Ser. No. 62/477,230, filed on Mar. 27, 2017, all of which areincorporated herein by reference in their entirety for all purposes.

STATEMENT OF GOVERNMENTAL SUPPORT

This invention was made with Government support under Grant NumberNS099800, awarded by the National Institutes of Health. The Governmenthas certain rights in the invention.

BACKGROUND

Cerebrospinal fluid (CSF) leak is a common complication of numerousprocedures in otolaryngology. It has been estimated that up to 13.8% ofendoscopic skull base surgeries result in CSF leaks. In the acutesetting, diagnostic options are limited. These include imagingtechniques such as magnetic resonance imaging (MRI) or computertomography (CT) or taking the patient directly to the operating room formanagement. This is largely guided by the surgeon's discretion andclinical intuition. These imaging modalities are costly and some exposethe patient to radiation. They also do not always identify a site ofleak when one exists. Alternatively, operative management is also anexpensive process that exposes the patient to general anesthesia andother perioperative risks. This involves identifying the site of theleak and using either native tissue or biocompatible materials to patchthe affected site.

Clinicians and surgeons need a quick, reliable and affordable test tohelp identify CSF leaks in the outpatient or postoperative settings. Itis often difficult to distinguish normal nasal and otologic secretionsfrom those containing CSF as they may be similar in appearance. Thisdistinction remains important because failure to recognize and repair aleak can result in severe complications, such as meningitis, brainstemherniation, and death. Currently, there are no proven, available teststhat allow a physician concerned about a CSF leak to inexpensively andnon-invasively rule out the presence of a leak. Alternative methods havebeen developed for the detection of CSF leaks, such as beta-2transferrin electrophoresis or enzyme-linked immunosorbent assay(ELISA), however they are rarely used due to high cost and longtime-to-result (often multiple days). If a patient has a CSF leak, atest that takes days to return is far too long. The patient can developmeningitis or brain herniation in that amount of time.

SUMMARY

There is a great and urgent clinical need for a quick, reliable andaffordable test to help identify CSF leaks in the outpatient orpostoperative settings. Recently, researchers have looked into thedetection of beta-trace protein (βTP) using a nephelometric assay andfound it to be comparable to beta-2 transferrin in sensitivity andspecificity for CSF. Although used in a research setting, this techniquehas yet to be utilized in the clinical setting.

In various embodiments devices and methods for determining the presence(or absence) of a cerebrospinal fluid (CSF) leak are provided. Incertain embodiments the devices utilize a semi-quantitative,barcode-style lateral-flow immunoassay (LFA) that quantifies the levelof beta-trace protein (βTP) in a sample.

Various embodiments contemplated herein may include, but need not belimited to, one or more of the following:

Embodiment 1: A lateral flow device for the semi-quantitative detectionof a cerebrospinal fluid leak, said device comprising:

a porous substrate;

a sample addition zone disposed on or in said porous substrate; adetection zone disposed on or in said porous substrate where saiddetection zone comprises at least a first test line (T1) and a secondtest line (T2) each test line comprising binding moieties that bind acomplex formed between beta-trace protein (βTP) and an indicatorattached to a βTP binding molecule;

wherein said porous substrate defines a flow path through which a sampleapplied to the sample addition zone flows under capillary action awayfrom said sample addition zone into said detection zone; and wherein

said first test line (T1) and said second test line (T2) are isconfigured so that either no test line signal or just a signal at thefirst test line (T1) is detectable when βTP concentration in a sampleapplied to said device is lower than the βTP level indicative of a CSFleak; and

said second test line (T2) is configured so that a signal is detectableat said second test line when βTP concentration in a sample applied tosaid device is greater than the βTP level indicative of a CSF leak.

Embodiment 2: The device of embodiment 1, wherein said first test line(T1) and said second test line (T2) are configured so that either notest line signal or just a signal at said first test line (T1) isdetectable when βTP concentration in a sample applied to said device islower than about 1.3 mg/L in a nasal drip sample.

Embodiment 3: The device of embodiment 2, wherein said first test line(T1) is configured so that a signal is detectable at said first testline when βTP concentration in a sample applied to said device isgreater than about 0.7 mg/L, or greater than about 0.8 mg/L, or greaterthan about 0.9 mg/L, or greater than about 1.0 mg/L, or greater thanabout 1.1 mg/L, or greater than about 1.2 mg/L, or at or greater than1.3 mg/L in a nasal drip sample.

Embodiment 4: The device according to any one of embodiments 1-3,wherein said first test line (T1) and said second test line (T2) areconfigured so that a signal is detectable at both test lines when βTPconcentration in a sample applied to said device is greater than about1.3 mg/L.

Embodiment 5: A lateral flow device for the semi-quantitative detectionof a cerebrospinal fluid leak, said device comprising:

a porous substrate;

a sample addition zone disposed on or in said porous substrate; adetection zone disposed on or in said porous substrate where saiddetection zone comprises at least a first test line (T1), a second testline (T2), and a third test line (T3), each test line comprising bindingmoieties that bind a complex formed between beta-trace protein (βTP) andan indicator attached to a βTP binding molecule;

wherein said porous substrate defines a flow path through which a sampleapplied to the sample addition zone flows under capillary action awayfrom said sample addition zone into said detection zone; and wherein

said first test line (T1) said second test line (T2) and said third testline (T3) are configured so that either no test line signal or just asignal at said first test line (T1) is detectable when βTP concentrationin a sample applied to said device is lower than or equal to the βTPlevel indicative of the absence of a CSF leak;

said first test line (T1) said second test line (T2) and said third testline (T3) are configured so that a signal is detectable at said firsttest line and said second test line, but not at said third test linewhen βTP concentration in a sample applied to said device is greaterthan the βTP level indicative of no CSF leak and less than a βTP leveldefinitive of the presence of a CSF leak; and

a signal is detectable at all three test lines T1, T2, and T3 when βTPconcentration in a sample applied to said device is equal to or greaterthan a βTP level definitive of the presence of a CSF leak.

Embodiment 6: The device of embodiment 5, wherein said first test line(T1) said second test line (T2) and said third test line (T3) areconfigured so that no signal is detectable at test lines T1, T2, or T3,or a signal is detectable only at test line T1, when βTP concentrationis less than about 0.7 mg/L in a nasal drip sample applied to saiddevice.

Embodiment 7: The device according to any one of embodiments 5-6,wherein said first test line (T1) said second test line (T2) and saidthird test line (T3) are configured so a signal is detectable at testlines T1, and T2, but not at test line T3, when βTP concentration rangesfrom about 0.7 mg/L up to about 1.3 mg/L in a nasal drip sample appliedto said device.

Embodiment 8: The device according to any one of embodiments 5-7,wherein said first test line (T1) said second test line (T2) and saidthird test line (T3) are configured so a signal is detectable at testlines T1, T2, and T3, when βTP concentration is greater than about 1.3mg/L in a nasal drip sample applied to said device.

Embodiment 9: The device according to any one of embodiments 1-8,wherein said lateral flow device comprise a control line downstream fromsaid first test line and said second test line, wherein said controlline comprises binding moieties that secondary antibody that can capturethe said indicator, in the presence or absence of βTP.

Embodiment 10: The device according to any one of embodiments 1-9,wherein said lateral flow device comprises a conjugation zone comprisingsaid indicator attached to a βTP binding molecule, where saidconjugation zone is disposed in said sample addition zone and/or betweensaid sample addition zone and said detection zone, wherein saidconjugation zone contains said indicator attached to a βTP bindingmolecule.

Embodiment 11: The device according to any one of embodiments 1-10,wherein said lateral flow device comprises an absorbent pad disposeddownstream from said detection zone and when said control line ispresent said absorbent pad is disposed downstream from said controlline.

Embodiment 12: The device according to any one of embodiments 1-11,wherein said porous substrate is disposed on a backing.

Embodiment 13: The device of embodiment 12, wherein said backingsubstantially fluid impermeable.

Embodiment 14: The device according to any one of embodiments 1-13,wherein said porous substrate is disposed in a cassette that provides aprotective covering and permits delivery of a sample to said sampleaddition zone, and visualization of the test lines in the detectionzone, and when present visualization of the control line.

Embodiment 15: The device according to any one of embodiments 1-14,wherein said indicator is selected from the group consisting of acolorimetric indicator, a fluorescent indicator, a radioactiveindicator, and a magnetic indicator.

Embodiment 16: The device of embodiment 15, wherein said indicator is acolorimetric indicator.

Embodiment 17: The device of embodiment 16, wherein said indictorcomprises a colorimetric indicator selected from the group consisting ofa dye, a nanoparticle, and a quantum dot.

Embodiment 18: The device of embodiment 17, wherein said indictorcomprises a gold nanoparticle.

Embodiment 19: The device according to any one of embodiments 1-18,wherein said βTP binding molecule comprises a moiety selected from thegroup consisting of an antibody, a lectin, a protein, a glycoprotein, anucleic acid, a small molecule, a polymer, a lipid, and combinationsthereof.

Embodiment 20: The device of embodiment 19, wherein said βTP bindingmolecule is an antibody or antibody fragment.

Embodiment 21: The device of embodiment 20, wherein said βTP bindingmolecule is an antibody that specifically binds βTP.

Embodiment 22: The device according to any one of embodiments 1-21,wherein said binding moieties that bind a complex formed betweenbeta-trace protein (βTP) and an indicator comprise a moiety selectedfrom the group consisting of an antibody, a lectin, a protein, aglycoprotein, a nucleic acid, a small molecule, a polymer, a lipid, andcombinations thereof.

Embodiment 23: The device of embodiment 22, wherein said bindingmoieties that bind a complex formed between beta-trace protein (βTP) andan indicator is an antibody or antibody fragment.

Embodiment 24: The device of embodiment 23, wherein said bindingmoieties that bind a complex formed between beta-trace protein (βTP) andan indicator is an antibody that specifically binds βTP.

Embodiment 25: The device according to any one of embodiments 1-24,wherein said device is configured to perform a sandwich assay.

Embodiment 26: The device according to any one of embodiments 1-25,wherein said porous substrate comprise a material selected from thegroup consisting of sintered glass or sintered ceramic, a mineral,cellulose, a fiberglass, a nitrocellulose, polyvinylidene fluoride, anylon, a charge modified nylon, a polyethersulfone, and combinationsthereof.

Embodiment 27: The device of embodiment 26, wherein said poroussubstrate comprises nitrocellulose.

Embodiment 28: A method of determining the presence of a cerebrospinalfluid (CSF) leak in a subject, said method comprising: applying abiological sample obtained from said subject to a the sample applicationzone of a device according to any one of clams 1-27; operating saiddevice in a lateral-flow assay format and detecting a signal if presentat test lines T1 and T2 in a device comprising two test lines and attest lines T1, T2, and T3 in a device comprising three test lines;and\where there is no detectable signal at any test line or a detectablesignal at only at test line T1, identifying the subject as a subjecthaving no CSF leak; where the device comprises two test lines and thereis a detectable signal at both test lines T1 and T2 identifying thesubject as having a CSF leak; where the device comprises three testlines and there is a detectable signal at all three test lines T1, T2,and T3, identifying the subject as having a CSF leak; where the devicecomprises three test lines and there is a detectable signal only twotest lines T1 and T2, identify the subject as in a indeterminate withrespect to CSF leaks and requiring further follow up.

Embodiment 29: The method of embodiment 28, wherein said device is alateral flow device containing two test lines.

Embodiment 30: The method of embodiment 28, wherein said device is alateral flow device containing three test lines.

Embodiment 31: The method according to any one of embodiments 28-30,wherein said device does not comprise a conjugation zone and saidindicator is combined with said sample before application to saiddevice.

Embodiment 32: The method according to any one of embodiments 28-30,wherein said device comprises a conjugation zone and said sample isapplied to said device, without addition of said indicator to saidsample.

Embodiment 33: The method according to any one of embodiments 28-32,wherein said sample is diluted prior to application to said device.

Embodiment 34: The method according to any one of embodiments 28-33,wherein said sample is diluted with phosphate-buffered saline (PBS).

Embodiment 35: The method according to any one of embodiments 28-34,wherein said subject is a human.

Embodiment 36: The method according to any one of embodiments 28-34,wherein said subject is a non-human mammal.

Embodiment 37: The method according to any one of embodiments 28-36,wherein said subject is a post-surgical subject.

Embodiment 38: The method of embodiment 37, wherein said subject has hadscalp or neurosurgery.

Embodiment 39: The method according to any one of embodiments 28-38,wherein said biological sample comprise a nasal drip sample and/or aserum sample.

Embodiment 40: The method according to any one of embodiments 28-39,wherein said method comprises determining βTP in a nasal drip samplefrom said subject and determining βTP in a serum sample of said subject.

Embodiment 41: The method of embodiment 40, wherein said methodcomprises determining the nasal drip to serum βTP concentration ratio.

Embodiment 42: The method of embodiment 41, wherein a ratio of <2 isclassified as negative for the presence of CSF and a ratio of >2 isclassified as positive for a CSF leak.

Embodiment 43: A kit for semi-quantification of βTP in a biologicalsample, said kit comprising a container containing a lateral flow devicefor the semi-quantitative detection of a cerebrospinal fluid leakaccording to any one of embodiments 1-27.

Embodiment 44: The kit of embodiment 43, wherein said kit contains twobarcode-style LFA test devices, one for testing serum and the other fortesting a nasal drip sample.

Embodiment 45: The kit according to any one of embodiments 43-44,wherein said kit comprises a reference card that correlates the numberand intensity of visible test lines with the concentrations of βTP inthe sample.

Embodiment 46: The kit according to any one of embodiments 43-45,wherein said kit further comprise a smartphone LSA reader for saiddevice.

Embodiment 47: The kit according to any one of embodiments 43-46,wherein said kit comprises instructions for using said kit forsemi-quantification of βTP in a biological sample.

Embodiment 48: A smartphone-based LFA reader for reading the test lineson a device according to any one of embodiments 1-27.

Embodiment 49: The reader of embodiment 48, wherein said reader isconfigured to photograph the test strips on said lateral flow assaydevice in a controlled light setting.

Embodiment 50: The reader of embodiment 49, wherein said device convertssaid photographs the photos to grayscale, locates the test lines on theLFA device by searching for peaks in pixel intensity.

Embodiment 51: The reader of embodiment 50, wherein said devicedetermines the mean or median grayscale intensity of each line and,optionally, the sum of all test line intensities.

Embodiment 52: The reader of embodiment 51, wherein said device comparesthe measured line intensities with an internal standard curve anddisplays the concentration of βTP in the sample and/or a clinicaldiagnosis.

Definitions

The terms “subject,” “individual,” and “patient” may be usedinterchangeably and typically a mammal, in certain embodiments a humanor a non-human primate. While the devices and methods are describedherein with respect to use in humans, they are also suitable for animal,e.g., veterinary use. Thus certain illustrative organisms include, butare not limited to humans, non-human primates, canines, equines,felines, porcines, ungulates, lagomorphs, and the like. Accordingly,certain embodiments contemplate the devices and methods described hereinfor use with domesticated mammals (e.g., canine, feline, equine),laboratory mammals (e.g., mouse, rat, rabbit, hamster, guinea pig), andagricultural mammals (e.g., equine, bovine, porcine, ovine), and thelike. The term “subject” does not require one to have any particularstatus with respect to a hospital, clinic, or research facility (e.g.,as an admitted patient, a study participant, or the like). Accordingly,in various embodiments, the subject can be a human (e.g., adult male,adult female, adolescent male, adolescent female, male child, femalechild) under the care of a physician or other health worker in ahospital, psychiatric care facility, as an outpatient, or other,clinical context. In certain embodiments, the subject may not be underthe care or prescription of a physician, or other, health worker.

As used herein, an “antibody” refers to a protein consisting of one ormore polypeptides substantially encoded by immunoglobulin genes orfragments of immunoglobulin genes. The recognized immunoglobulin genesinclude the kappa, lambda, alpha, gamma, delta, epsilon and mu constantregion genes, as well as myriad immunoglobulin variable region genes.Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

A typical immunoglobulin (antibody) structural unit is known to comprisea tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains, respectively.

Antibodies exist as intact immunoglobulins or as a number of wellcharacterized fragments produced by digestion with various peptidases.Thus, for example, pepsin digests an antibody below the disulfidelinkages in the hinge region to produce F(ab)′₂, a dimer of Fab whichitself is a light chain joined to V_(H)-C_(H)1 by a disulfide bond. TheF(ab)′₂ may be reduced under mild conditions to break the disulfidelinkage in the hinge region thereby converting the (Fab′)₂ dimer into aFab′ monomer. The Fab′ monomer is essentially a Fab with part of thehinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press,N.Y. (1993), for a more detailed description of other antibodyfragments). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchFab′ fragments may be synthesized de novo either chemically or byutilizing recombinant DNA methodology. Thus, the term antibody, as usedherein also includes antibody fragments either produced by themodification of whole antibodies or synthesized de novo usingrecombinant DNA methodologies. Certain preferred antibodies includesingle chain antibodies (antibodies that exist as a single polypeptidechain), more preferably single chain Fv antibodies (sFv or scFv) inwhich a variable heavy and a variable light chain are joined together(directly or through a peptide linker) to form a continuous polypeptide.The single chain Fv antibody is a covalently linked V_(H)-V_(L)heterodimer which may be expressed from a nucleic acid including V_(H)-and V_(L)-encoding sequences either joined directly or joined by apeptide-encoding linker. Huston, et al. (1988) Proc. Nat. Acad. Sci.USA, 85: 5879-5883. While the V_(H) and V_(L) are connected to each as asingle polypeptide chain, the V_(H) and V_(L) domains associatenon-covalently. The first functional antibody molecules to be expressedon the surface of filamentous phage were single-chain Fvs (scFv),however, alternative expression strategies have also been successful.For example Fab molecules can be displayed on phage if one of the chains(heavy or light) is fused to g3 capsid protein and the complementarychain exported to the periplasm as a soluble molecule. The two chainscan be encoded on the same or on different replicons. The importantpoint is that the two antibody chains in each Fab molecule assemblepost-translationally and the dimer is incorporated into the phageparticle via linkage of one of the chains to, e.g., g3p (see, e.g., U.S.Pat. No. 5,733,743). The scFv antibodies and a number of otherstructures converting the naturally aggregated, but chemically separatedlight and heavy polypeptide chains from an antibody V region into amolecule that folds into a three dimensional structure substantiallysimilar to the structure of an antigen-binding site are known to thoseof skill in the art (see e.g., U.S. Pat. Nos. 5,091,513, 5,132,405 and4,956,778). Particularly preferred antibodies should include all thathave been displayed on phage (e.g., scFv, Fv, Fab and disulfide linkedFv) (Reiter et al. (1995) Protein Eng. 8: 1323-1331).

The term “biological sample” refers to sample is a sample of biologicaltissue, cells, or fluid that, in a healthy and/or pathological state, ismay contain CSF in a subject that has a CSF leakage. Illustrativesamples include, but are not limited to blood or plasma samples, nasaland/or oral fluid samples, and the like. Although the sample istypically taken from a human patient, the assays can be used to detectCSF in samples from any mammal, such as dogs, cats, sheep, cattle, andpigs, etc. The sample may be pretreated as necessary by dilution in anappropriate buffer solution or concentrated, if desired. Any of a numberof standard aqueous buffer solutions, employing one of a variety ofbuffers, such as phosphate, Tris, or the like, at physiological pH canbe used and the term sample is intended to include pre-treated samplesas well as acute samples.

The phrase a “signal is detectable at a test line” or detectable at acontrol line means a signal indicative of the present of the moiety ofinterest at the test line. For example, where the signal is acolorimetric signal the signal is detectable if visible with the nakedeye or detected using a reader device for the lateral flow assay.

The term “downstream” when used with reference to a lateral flow deviceindicates that the downstream location is further along the lateral flowdevice in the direction of fluid flow (capillary flow) than anotherlocation. Thus, if location 2 is downstream from location 1, then fluidflowing through the lateral flow device will reach location 1 beforereaching location 2.

As used herein, the term “instructions for using said kit forsemi-quantification of βTP in a biological sample” includes instructionsfor using the reagents contained in the kit for the detection and/orsemiquantification of βTP. In some embodiments, the instructions furthercomprise the statement of intended use required by the U.S. Food andDrug Administration (FDA) in labeling in vitro diagnostic products. TheFDA classifies in vitro diagnostics as medical devices and requires thatthey be approved through the 510 (k) procedure. Information required inan application under 510 (k) includes: 1) The in vitro diagnosticproduct name, including the trade or proprietary name, the common orusual name, and the classification name of the device; 2) The intendeduse of the product; 3) The establishment registration number, ifapplicable, of the owner or operator submitting the 510 (k) submission;the class in which the in vitro diagnostic product was placed undersection 513 of the FD&C Act, if known, its appropriate panel, or, if theowner or operator determines that the device has not been classifiedunder such section, a statement of that determination and the basis forthe determination that the in vitro diagnostic product is not soclassified; 4) Proposed labels, labeling and advertisements sufficientto describe the in vitro diagnostic product, its intended use, anddirections for use. Where applicable, photographs or engineeringdrawings should be supplied; 5) A statement indicating that the deviceis similar to and/or different from other in vitro diagnostic productsof comparable type in commercial distribution in the U.S., accompaniedby data to support the statement; 6) A 510 (k) summary of the safety andeffectiveness data upon which the substantial equivalence determinationis based; or a statement that the 510 (k) safety and effectivenessinformation supporting the FDA finding of substantial equivalence willbe made available to any person within 30 days of a written request; 7)A statement that the submitter believes, to the best of their knowledge,that all data and information submitted in the premarket notificationare truthful and accurate and that no material fact has been omitted; 8)Any additional information regarding the in vitro diagnostic productrequested that is necessary for the FDA to make a substantialequivalency determination. Additional information is available at theInternet web page of the U.S. FDA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a schematic of a typical lateral-flow immunoassaytest strip (top). The sandwich format of the lateral-flow immunoassay isillustrated in the bottom panel.

FIG. 2 illustrates a schematic and illustrative βTP cutoff levels of atwo-line LFA design.

FIG. 3 illustrates dilutions of human CSF in human serum (HS). Negativecontrol, HS, displays 1 visible test line. All tests containing CSFdisplay 2 test lines. There were no control lines printed on thesestrips.

FIG. 4 shows a schematic and illustrative βTP cutoff levels of athree-test line LFA design.

FIG. 5 schematically illustrates a lateral-flow immunoassayincorporating a conjugation zone.

DETAILED DESCRIPTION

In various embodiments devices and methods are provided for the rapiddetection of cerebrospinal fluid (CSF) leaks in a subject. The devicesand methods provide a rapid, robust, easy-to-use, and inexpensivepoint-of-care diagnostic device that can distinguish between samplescontaining CSF and those that do not.

The current standards for diagnosing CSF leaks include invasive andexpensive techniques such as CT and MM, as well as ELISA and beta-2transferrin electrophoresis, which are rarely used due to their highcost and long time-to-result. More recently, researchers have lookedinto the detection of beta-trace protein (βTP) using a nephelometricassay. While rapid and highly sensitive, the βTP nephelometric assaystill requires expensive equipment found in a centralized laboratory andextensive training.

The devices described herein provide the ability to rapidly quantify theconcentration of βTP in a sample in order to determine the presence orabsence of a CSF with the use of a barcode-style lateral flow assay(LFA). This technology can allow for the rapid diagnosis of a CSF leakwithout the need for expensive equipment, invasive tests, or extensivetraining. Moreover the devices and methods facilitate rapid detection ofa CSF leak before the subject decompensates as a consequence of theleakage.

Accordingly the methods and devices described herein are directed to thedetection of βTP in a subject (e.g., in a biological sample derived froma subject). In particular embodiments, lateral-flow assay devices (e.g.,bar-code lateral flow devices) are provided for the detection andsemi-quantitative determination of CSF in a sample thereby permittingthe diagnosis of CSF leaks in a subject. It is noted that, to the extentthat the following description is of a specific embodiment or aparticular uses, it is intended to be illustrative only, and notlimiting of the claimed invention. The following description is intendedto cover all alternatives, modifications and equivalents that areincluded in the spirit and scope of the invention, as defined in theclaims.

Lateral flow immunoassays are simple tests for rapid detection of thepresence or absence of a target analyte in a sample for home testing,point of care testing, or laboratory applications. “Bar-code” or“ladder” lateral flow immunoassay provides a semi-quantitativemeasurement of the amount of analyte present in a sample typically byproviding multiple detection bands (e.g., in a detection zone) zone eachof which represents a different analyte concentration range.

Lateral flow test strips typically utilize a solid support through whicha mobile phase (e.g., a liquid sample) can flow through by capillaryaction to a reaction matrix where a detectable signal, such as colorchanges or color differences on the test strip, may be generated toindicate the presence or absence of the target analyte. As used herein,the term “capillary action” or “capillarity” means the process by whicha molecule is drawn across the lateral test strip due to such propertiesas surface tension and attraction between molecules.

In certain embodiments the concentration of βTP in a sample isquantified using a barcode-style lateral-flow immunoassay (LFA) in orderto detect (e.g., determine the presence of or absence of, or severityof) a CSF leak. A typical LFA consists of at least 3 components: asample pad where the sample is applied to the test strip, a detectionzone where there is binding and where results can be observed, and anabsorbent pad which acts as a sink for excess sample (FIG. 1, top). Incertain embodiments of a sandwich assay format, the LFA indicator (whichcan be colorimetric, fluorescent, radioactive, etc.) decorated withbinding molecules (e.g., antibodies, aptamers, etc.) can be first addedto the sample which is then applied to the LFA device. Alternatively, incertain embodiments the LFA comprise a conjugation zone containing theindicator attached to binding molecules and combination of the samplewith the indicator can occur in the conjugation zone. If the target, inthis case βTP, is present, it will bind to the indicator decorated withthe binding molecule. These complexes are flow through the strip intothe detection zone towards the absorbent pad when present. If βTP ispresent, they associate with the binding molecules immobilized on thetest line(s) and become sandwiched between the indicator and themembrane providing a signal at the particular test line(s). For example,if the indicator is colorimetric, the colorimetric indicator willexhibit a strong color, and a visual band forms as the βTP-indicatorcomplex accumulates at the test line, indicating a positive result.Alternatively, if no βTP is present, the indicator does not attach tothe test line, and the absence of the test line indicates a negativeresult. Regardless of the presence of βTP, the binding moleculedecorated on the indicator can associate with and accumulate on thecontrol line when a control line is present. A band at the control linesignifies that the sample has flowed through the strip, indicating avalid test. In certain embodiments then, a positive result is thereforeindicated by two bands, one at the test line and one at the controlline, while a negative result is indicated by a single band at thecontrol line (FIG. 1, bottom).

While the traditional LFA gives a qualitative “yes” or “no” readout, amore quantitative device would be instrumental in the accurate diagnosisof a CSF leak, where lower concentrations of βTP are indicative of anegative result, and higher concentrations of βTP are indicative of apositive result for a CSF leak. An alternative format of the LFA thatprovides a more quantitative output is called the barcode-style orladder LFA, in which multiple test lines are printed on the detectionzone. Each test line will have a cut-off, which is the minimumconcentration of βTP in a sample necessary for that test line to becomevisible. These cut-offs can be adjusted by varying the density of thebinding molecule that is immobilized, as well as altering the affinitythat binding molecule has for βTP. After running the assay, the numberof test lines that are visible on the LFA strip can be correlated withan approximate βTP concentration in the sample and thus a clinicaldiagnosis.

Accordingly, in certain embodiments, a semi-quantitative LFA assay isprovided with two test lines to quantify βTP levels. To develop the twotest line assay, an antibody to βTP, the capture antibody, isimmobilized on the detection zone of the LFA membrane in two locations:test line 1 (T1), and test line 2 (T2). Previous studies have shown thata βTP concentration of >1.3 mg/L, in a nasal drip sample, is a goodindicator of a CSF leak.

Accordingly, in certain embodiments, capture moieties (e.g., captureantibodies) are immobilized on the detection zone such that 0 or 1 testline will be detectable (e.g., visible) when the concentration of βTP isbelow 1.3 mg/L and 2 test lines will be detectable (e.g., visible) whenthe concentration of βTP is above 1.3 mg/L (FIG. 2). A secondaryantibody that can capture the colorimetric indicator, in the presence orabsence of βTP, can be immobilized as the control line, downstream fromthe test lines. In the following example of the two test linebarcode-style LFA, gold nanoparticles decorated with anti-βTP antibodies(GNPs) were used as the colorimetric indicator.

In certain embodiments prior to running the assay, the sample isdiluted, e.g. 300-fold in phosphate-buffered saline (PBS). The dilutedsample is mixed with GNPs and is then applied to the sample pad, whereit will begin to flow through the LFA strip. Any βTP in the sample willbind to the GNPs to form βTP-GNP complexes. When these βTP-GNP complexescross the detection zone, they will first bind to T1. βTP-GNP complexesthat do not bind to T1 will be able to bind to T2. Finally, unbound GNPsand any remaining βTP-GNP complexes will bind to the control line toindicate a valid test. After, e.g., 20 minutes, the number of detectable(e.g., visible) test lines is counted. If 0 or 1 test line is present,the sample is considered negative for a CSF leak. If 2 test lines arepresent, the sample is considered positive for a CSF leak.

To demonstrate the two-test line LFA device, a CSF sample obtained froma lumbar drain was diluted in human serum to simulate a clinical nasaldrip sample containing trace amounts of CSF. When run on the 2 test lineLFA device, all samples containing CSF displayed 2 visible test lines,indicating positive results for the presence of CSF. When the negativecontrol, human serum without CSF, was run, only one test line appeared,indicating a negative result for the presence of CSF (FIG. 3). Controllines were not printed in the results shown.

In another illustrative, but non-limiting embodiments, asemi-quantitative LFA device with three test lines to quantify βTPlevels is provided. A βTP concentration of >1.3 mg/L has been shown tohave relatively high sensitivity when detecting for CSF leaks using thenephelometric assay. However, in the region from 0.7-1.3 mg/L, which wewill refer to as a “grey zone”, it has been reported that approximately1 in 3 samples will contain CSF. As a result, if all βTP concentrationsof <1.3 mg/L are classified as negative for CSF, potentiallylife-threatening CSF leaks will be misdiagnosed. Therefore, it isbeneficial to develop a device that can identify samples in this greyzone, so that further testing can be performed before ruling out thepresence of a CSF leak. To develop this assay, an antibody to βTP, thecapture antibody, is immobilized on the detection zone of the LFAmembrane in three locations: test line 1 (T1), test line 2 (T2), andtest line 3 (T3). The capture moieties (e.g., antibodies) will beimmobilized such that 0 or 1 test line will be visible when theconcentration of βTP in a sample is below 0.7 mg/L, 2 test lines will bevisible when the concentration is 0.7-1.3 mg/L, and 3 test lines will bevisible when the concentration is greater than 1.3 mg/L (FIG. 4). Asecondary antibody, that can capture the indicator bound with detectionantibodies, in the presence or absence of βTP, is immobilized as thecontrol line, downstream from the test lines. In the following exampleof the three test line barcode-style LFA, gold nanoparticles decoratedwith anti-βTP antibodies (GNPs) are used as the colorimetric indicator.

In certain embodiments prior to running the assay, the sample to be runis diluted, e.g., 300-fold in phosphate-buffered saline (PBS). Thediluted sample is then applied to the sample pad, where it begins toflow through the LFA strip. Any βTP in the sample binds to the GNPs toform βTP-GNP complexes. When these βTP-GNP complexes cross the detectionzone, they will first bind to T1. βTP-GNP complexes that do not bind toT1 will be able to bind to T2. βTP-GNP complexes that do not bind to T1or T2 will then be able to bind to T3. Finally, the unbound GNPs and anyremaining βTP-GNP complexes will bind to the control line to indicate avalid test. After, e.g., 20 minutes, the number of colored test lines iscounted. If 0 or 1 test line is present, the sample is considerednegative for a CSF leak. If 2 test lines are present, the sample isconsidered in the grey zone, where further testing is required and it isrecommended to rerun the assay with a newly collected sample. If 3 testlines are present, the sample is considered positive for a CSF leak.

Another aspect of this invention is the development of a kit thatcontains two separate LFA tests to quantify the level of βTP in both apatient's serum and nasal drip sample. Previous studies havedemonstrated that the ratio of βTP concentrations in nasal drip sampleto serum is also a sensitive and specific indicator of the presence orabsence of a CSF leak. This invention looks to use the barcode-style LFAto quantify the βTP concentration in both serum and nasal drip samples.In certain embodiments the kit will contain two barcode-style LFA testdevices, one for testing serum and the other for testing the nasal dripsample. In certain embodiments it can also contain a reference card thatcorrelates the number and intensity of visible test lines with theconcentrations of βTP in the sample. After running a patient's serum andnasal drip samples on their respective LFA test strips and obtaining theβTP concentrations in each samples, the nasal drip to serum βTPconcentration ratio will be taken. In certain embodiments a ratio ofless than 2 (<2) is classified as negative for the presence of CSF and aratio of greater than 2 (>2) or greater than or equal to 2 is positive.

Other embodiments contemplated herein involve the integration of asmartphone-based LFA reader with the LFA test strips described above. Anoptional smartphone-based LFA reader can further improve theease-of-interpretation and accuracy of the tests for the detection ofCSF leaks. Photographs of the LFA test strips are taken (in certainembodiments in a controlled light setting, such as an external photolight box or an integrated phone attachment that blocks outside lightand uses the smartphone flash as a lighting source). A smartphoneapplication then converts the photos to grayscale (e.g., 8-bit, 16-bit,32-bit grayscale). Each test line on the LFA strip can be located bysearching for peaks in pixel intensity. Once the location of each testline is determined, the mean grayscale intensity of each line can becalculated and/or as the sum of all test line intensities. The measuredintensities can be compared with an internal standard curve and theconcentration of βTP in the sample and/or the clinical diagnosis can bedisplayed on the smartphone screen.”

Lateral Flow Assay Components.

In various embodiments the lateral flow assay comprises a poroussubstrate, a sample addition zone disposed on or in the poroussubstrate; and a detection zone disposed on or in said porous substratewhere said detection zone comprises at least a first test line (T1) anda second test line (T2) and in certain embodiments a third test line(T3) as described above. In certain embodiments the lateral flow assayadditionally comprises a conjugation zone containing the indicatorattached to a moiety that binds βTP. The lateral flow device canadditionally comprise a control line and/or an absorbent pad (e.g.,sink).

Sample Receiving Zone

The LFA devices described herein typically comprise a sample receivingzone for application of the biological sample to the device. In certainembodiments the sample receiving zone comprise a sample pad disposed onor in the porous substrate. In certain embodiments the sample pad canact as a filter that can remove debris, contaminants, and mucus from thecollected fluid. It can also store dried reagents, and when rehydrated,these reagents can (i) adjust the solution for optimal detectionconditions (pH, ionic strength, etc); and (ii) break down mucus,glycoproteins, and other viscous materials in the collected specimenthat may affect detection. Illustrative materials for the sample padinclude, but are not limited to, cellulose, nitrocellulose, fiberglass,cotton, woven or nonwoven paper, etc. Reagents on the pad may include,but are not limited to, surfactants such as Triton X-100, Tween 20, orsodium dodecyl sulfate, etc.; polymers such as polyethylene glycol,poloxamer, polyvinylpyrrolidone (PVP), etc.; buffers such asphosphate-buffered saline, 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid (HEPES), Tris(hydroxymethyl)aminomethane (Tris), sodium borate,TRICINE, etc.; proteins such as albumin, etc.; enzymes such as protease,etc.; salts such as sodium chloride, sodium phosphate, sodium cholate,potassium phosphate, etc. In various embodiments these reagents can beapplied to the sample pad by (i) soaking the paper material in thereagent solution, or (ii) through wicking the membrane via capillaryflow. The treated sample pad can be dried by (i) air dry (let sit inroom temperature); (ii) baking (place in high temperature using an ovenor heating device); (iii) vacuum; or (iv) lyophilization.

Conjugation Zone

In certain embodiments the LFA devices described herein can comprise aconjugation zone for mixing the sample with an indicator attached to aβTP binding molecule (see, e.g., FIG. 5). In certain embodiments theconjugation zone comprises a conjugate pad. In certain embodiments theconjugation zone, when present can contain dehydrated indicators (e.g.,colorimetric indicators, a fluorescent indicators, a radioactiveindicators, magnetic indicators, etc.) decorated with binding moietiesthat bind the βTP target analyte. In certain embodiments the bindingmoieties are specific binding moieties that have high affinity towardsβTP. When the sample solution reaches the conjugate pad, the indicators(e.g., colorimetric indicators) are rehydrated. The binding moieties onthe indicators can then bind to the βTP the resulting complexes can flowto the detection zone. In certain embodiments the indicators cancomprise colorimetric indicators that can comprise metallic particlessuch as gold, silver particles, polymeric particles such as latex beads,and polystyrene particles encapsulating visible or fluorescent dyes.Illustrative materials material for the conjugation zone (e.g.,conjugate pad) include, but are not limited to, cellulose,nitrocellulose, fiberglass, cotton, woven or nonwoven paper etc. Incertain embodiments the colorimetric indicators can be applied anddehydrated onto the pad as described above.

Detection Zone

In certain embodiments the reaction pad, when present, can compriseimmobilized reagents, and when the immobilized reagents react with thesample solution, they may produce signals (e.g., visual signals) toindicate the presence or absence or quantity of the target analyte(s) atparticular test lines. Illustrative materials for the detection zoneinclude, but are not limited to cellulose, nitrocellulose, fiberglass,cotton, woven or nonwoven paper etc.

In certain embodiments for a lateral-flow test strip, the reagents inthe detection zone are immobilized in the form of lines perpendicular tothe direction of flow to ensure all samples can interact with theimmobilized reagents. The concentrations of the reagents can beoptimized to control the signal intensities, and thus, control thesensitivity of the assay. For example, a semi-quantitative assay can bedesigned by immobilizing multiple lines of the same reagent with variousconcentrations. Each line therefore will yield signals only when aspecific concentration of target biomolecules is reached. Theconcentration of the target biomolecules can then be interpreted bycounting the number of lines that are visible, e.g., as described above.

Absorbent Pad/Sink

In certain embodiments the lateral flow device comprises an absorbentpad disposed downstream from the detection zone and when said controlline is present the absorbent pad is disposed downstream from thecontrol line. In certain embodiments the sink, when present, cancomprise an absorbent pad that collect excess fluid and preventsback-flow which can affect the test performance. Illustrative materialsfor the sink include, but are not limited to cellulose, nitrocellulose,fiberglass, cotton, woven and nonwoven paper etc.

Lateral Flow Assay (LFA)or Flow-Through (Spot) Assay

As explained above, in certain embodiments, the devices and systemsdescribed herein are configured to provide a lateral flow assay (LFA)for detection of the target analyte (βTP) in a sample. The LFA typicallycomprises a porous matrix into which is disposed sample and assaycomponents, e.g., as described above. The porous matrix is configured toand has porosity sufficient to allow the assay reagents to flow throughthe porous matrix when the components are in a fluid phase. Such porousLFA devices are can be referred to as paper or paper fluidic devices andthese terms are used interchangeably.

The term “paper”, as used herein, is not limited to thin sheets from thepulp of wood or other fibrous plant substances although, in certainembodiments the use of such papers in the devices described herein iscontemplated. Papers more generally refer to porous materials often insheet form, but not limited thereto that allow a fluid to flow through.

In some embodiments, the porous matrix is sufficiently porous to allowthe assay components and target analyte(s) to flow through the LFA. Insome embodiments the porous matrix comprises inter alia a material suchas a scintered glass ceramic, a mineral, cellulose, a fiberglass, anitrocellulose, polyvinylidene fluoride, a nylon, a charge modifiednylon, a polyethersulfone, combinations thereof, and the like.

Sandwich Assay

In some embodiments, the LFA is configured to provide or run a sandwichassay (see e.g., FIG. 1, bottom left, in copending PCT Application No:PCT/US2015/019297, filed on Mar. 6, 2015, which is hereby incorporatedby reference for the LFA configurations described therein). In someembodiments, the sandwich assay comprises a capture moiety that bindsthe target analyte. In some embodiments, the device comprises anindicator attached to a moiety that binds to the analyte of interestβTP). In some embodiments, the indicator comprises a detectable property(colorimetric, fluorescent, radioactive, etc.). In some embodiments, theprobe comprises a binding moiety that interacts with the target analyte(e.g. an antibody). In some embodiments, the indicator is added to thesample before application to the device and binds the target analyte toform a probe-analyte complex. In some embodiments, the indicator iscombined with the sample in a conjugation zone in the LFA device afterthe sample is added to the device and binds the target analyte to form aprobe-analyte complex.

Indicator-analyte complex flows through the LFA or through theflow-through device towards the absorbent pad. In some embodiments, thetarget analyte of the indicator-analyte complex binds to the capturemoiety. In some embodiments, the capture moiety is immobilized on a testline or a test region (e.g., a test layer in a flow-through device) andthe indicator-analyte complex becomes immobilized on the test line or inthe test region. In some embodiments, the indicator is colorimetric, andthe test line or test region will exhibit a strong color (e.g.detectable signal) as the indicator-analyte complex accumulates at thetest line or in the test region, indicating a positive result. In someembodiments, there is no target analyte present in the sample, and theindicator of the indicator-analyte complex does not interact with thecapture moiety, and the absence of the test line or signal in the testregion indicates a negative result at that test line. In someembodiments, the LFA comprises an indicator capture moiety on a controlline (or in a control region, e.g., of a flow-through assay device) thatinteracts directly with the indicator and/or the binding moiety, andthus, regardless of the presence of the target analyte in the sample,the indicator/binding moiety binds to the probe capture moiety andaccumulates on the control line or in the control region. In someembodiments, the indicator capture moiety is a secondary antibody thatbinds the binding moiety, wherein the binding moiety is a primaryantibody that binds that target analyte. In some embodiments, theindicator becomes immobilized and detected on the control line or in thecontrol region, indicating a valid test. In some embodiments, a positiveresult (e.g. target analyte is present in sample) is indicated by adetectable signal at the test line(s) as described above. In someembodiments, a negative result is indicated by a detectable signal atthe control line or in the control region in the absence of test linesignal(s) as described above.

Indicators

In certain embodiments the systems and/or devices described hereinand/or the methods described herein utilize an indicator, where theindicator comprises a binding moiety that binds the target analyte toform an indicator-analyte complex.

In some embodiments, the indicator comprises a material selected fromthe group consisting of a synthetic polymer, a metal, a mineral, aglass, a quartz, a ceramic, a biological polymer, a plastic, andcombinations thereof. In some embodiments, the indicator comprises apolymer selected from the group consisting of polyethylene,polypropylene, nylon (DELRIN®), polytetrafluoroethylene (TEFLON®),dextran and polyvinyl chloride. In some embodiments, the polyethylene ispolyethylene glycol. In some embodiments, the polypropylene ispolypropylene glycol. In some embodiments, the indicator comprises abiological polymer selected from the group consisting of collagen,cellulose, and chitin. In some embodiments, the indicator comprises ametal selected from the group consisting of gold, silver, platinumtitanium, stainless steel, aluminum, and alloys thereof. In someembodiments, the indicator comprises a nanoparticle (e.g., a goldnanoparticle, a silver nanoparticle, etc.).

In some embodiments, the indicator comprises a detectable label.Detectable labels include any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical, orchemical means. Illustrative useful labels include, but are not limitedto, fluorescent nanoparticles (e.g., quantum dots (Qdots)), fluorescentdyes (e.g., fluorescein, texas red, rhodamine, green fluorescentprotein, and the like, see, e.g., Molecular Probes, Eugene, Oreg., USA),radiolabels (e.g., ³H, ¹²⁵I, ³⁵S,¹⁴C, ³²P, ⁹⁹Tc, ²⁰³Pb, ⁶⁷Ga, ⁶⁸Ga,⁷²As, ¹¹¹In, ^(113m)In, ⁹⁷Ru, ⁶²Cu, 641Cu, ⁵²Fe, ^(52m)Mn, ⁵¹Cr, ¹⁸⁶Re,¹⁸⁸Re, ⁷⁷As, ⁹⁰Y, ⁶⁷Cu, ¹⁶⁹Er, ¹²¹Sn, ¹²⁷Te, ¹⁴²Pr, ¹⁴³Pr, ¹⁹⁸Au, ¹⁹⁹Au,¹⁶¹Tb, ¹⁰⁹Pd, ¹⁶⁵Dy, ¹⁴⁹Pm, ¹⁵¹Pm, ¹⁵³Sm, ¹⁵⁷Gd, ¹⁵⁹Gd, ¹⁶⁶Ho, ¹⁷²Tm,¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁰⁵Rb, ¹¹¹Ag, and the like), enzymes (e.g., horseradish peroxidase, alkaline phosphatase and others commonly used in anELISA), various colorimetric labels, magnetic or paramagnetic labels(e.g., magnetic and/or paramagnetic nanoparticles), spin labels,radio-opaque labels, and the like.

Alternatively or additionally, the indicator can bind to anotherparticle that comprises a detectable label. In some embodiments, theindicator provide a detectable signal at the detection zone (e.g. testline, control line, test region, control region). In some embodiments,the detectable label/property is selected from the group consisting of acolorimetric label/property, a fluorescent label/property, an enzymaticlabel/property, a colorigenic label/property and a radioactivelabel/property. In some embodiments, the indicator comprises a goldnanoparticle and the detectable property is a color. In someembodiments, the color is selected from orange, red and purple.

In some embodiments, the indicator further comprises a coating. In someembodiments, the coating comprises polyethylene glycol or polypropyleneglycol. In some embodiments, the coating comprises polypropylene. Insome embodiments, the coating comprises polypropylene glycol. In someembodiments, the coating comprises dextran. In some embodiments, thecoating comprises a hydrophilic protein. In some embodiments, thecoating comprises serum albumin. In some embodiments, the coating has anaffinity for the first phase solution or the second phase solution.

Binding Moiety

In some embodiments, the binding moiety is a molecule that binds thetarget analyte (βTP). In some embodiments, the binding moiety is amolecule that specifically binds βTP. In some embodiments, “specificallybinds” indicates that the molecule binds preferentially to βTP or bindswith greater affinity to the target analyte than to other molecules. Byway of non-limiting example, an antibody will selectively bind to anantigen against which it was raised. Also, by way of non-limitingexample, a DNA molecule will bind to a substantially complementarysequence and not to unrelated sequences under stringent conditions. Insome embodiments, “specific binding” can refer to a binding reactionthat is determinative of the presence of a target analyte in aheterogeneous population of molecules (e.g., proteins and otherbiologics). In some embodiments, the binding moiety binds to itsparticular target analyte and does not bind in a significant amount toother molecules present in the sample.

In some embodiments, the binding moiety is selected from the groupconsisting of an antibody, a lectin, a protein, a glycoprotein, anucleic acid, monomeric nucleic acid, a polymeric nucleic acid, anaptamer, an aptazyme, a small molecule, a polymer, a lectin, acarbohydrate, a polysaccharide, a sugar, a lipid, and any combinationthereof. In some embodiments, the binding moiety is a molecule capablebinding pair the target analyte.

In some embodiments, the binding moiety is an antibody or antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)₂, Fv, Fv′, Fd, Fd′, scFv, hsFv fragments, single-chainantibodies, cameloid antibodies, diabodies, and other fragmentsdescribed herein.

In certain embodiments, the binding moiety comprises an aptamer. In someembodiments, the aptamer comprises an antibody-analogue formed fromnucleic acids. In some embodiments, the aptamer does not require bindingof a label to be detected in some assays, such as nano-CHEM-FET, wherethe reconfiguration would be detected directly. In some embodiments, thebinding moiety comprises an aptazyme. In some embodiments, the aptazymecomprises an enzyme analogue, formed from nucleic acids. In someembodiments, the aptazyme functions to change configuration to capture aspecific molecule, only in the presence of a second, specific, analyte.

Sample Collection

In various embodiments the sample to be assayed using the devices andmethods described herein comprises a biological sample. Illustrativebiological samples include, but are not limited to biofluids such asblood or blood fractions, lymph, nasal or oral fluids, and the like.

Where the biological sample comprises a tissue, in certain embodiments,the tissue may be lysed, homogenized, and/or ground and, optionallysuspended in a sample solution. Where the biological sample comprise abiological fluid the fluid may be assayed directly or suspended in asample solution prior to assay. In certain embodiments the samplesolution may act to preserve or stabilize the biological sample orcomponents thereof, and/or may act to extract or concentrate thebiological sample or components thereof. In certain embodiments thesample solution may comprise a buffer, optionally containingpreservatives, and/or enzymes (protease, nuclease, etc.), and/orsurfactants, and/or ATPS components.

In certain embodiments, particular in point-of-care embodiments, thesample may be applied to the assay device immediately or after a modesttime interval. In certain embodiments the sample may be delivered to aremote testing facility where the assay is run.

Methods and devices for collecting biological samples are well known tothose of skill in the art.

Kits.

In certain embodiments a kit for semi-quantification of βTP in abiological sample is provided. In certain embodiments the kits comprisea container containing a lateral flow device for the semi-quantitativedetection of a cerebrospinal fluid leak as described herein. In certainembodiments the kit contains two barcode-style LFA test devices asdescribed herein, one for testing serum and the other for testing anasal drip sample. In certain embodiments the kit comprises a referencecard that correlates the number and intensity of visible test lines withthe concentrations of βTP in the sample. In certain embodiments the kitfurther comprises a smartphone LSA reader for the LSA device(s). Incertain embodiments the kit comprises one or more sample collectiondevices (e.g., devices for collecting blood or plasma, or nasal or oralmaterial).

In certain embodiments the kit comprises instructions (instructionalmaterials) for using the kit for semi-quantification of βTP in abiological sample.

While the instructional materials typically comprise written or printedmaterials, they are not limited to such. Any medium capable of storingsuch instructions and communicating them to an end user is contemplatedby this invention. Such media include, but are not limited to,electronic storage media (e.g., magnetic discs, tapes, cartridges,chips), optical media (e.g., CD ROM), and the like. Such media mayinclude addresses to internet sites that provide such instructionalmaterials.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. A smartphone-based lateral flow assay (LFA)reader configured to read test lines on a lateral flow device fordetecting a cerebrospinal fluid leak, where said lateral flow devicecomprises: a porous substrate; a sample addition zone disposed on or insaid porous substrate; a detection zone disposed on or in said poroussubstrate where said detection zone comprises at least a first test line(T1) and a second test line (T2) each test line comprising bindingmoieties that bind a complex formed between beta-trace protein (βTP) andan indicator attached to a βTP binding molecule; wherein said poroussubstrate defines a flow path through which a sample applied to thesample addition zone flows under capillary action away from said sampleaddition zone into said detection zone; and wherein: said first testline (T1) and said second test line (T2) are configured so that eitherno test line signal or just a signal at the first test line (T1) isdetectable when βTP concentration in a sample applied to said device islower than the βTP level indicative of a cerebrospinal fluid (CSF) leak;and said second test line (T2) is configured so that a signal isdetectable at said second test line when βTP concentration in a sampleapplied to said device is greater than the βTP level indicative of a CSFleak.
 2. The smartphone-based LFA reader of claim 1, wherein said readeris configured to photograph the test lines on said lateral flow devicein a controlled light setting to produce photographs of said test lines.3. The smartphone-based LFA reader of claim 2, wherein said readerconverts said photographs to grayscale, and locates the test lines onthe LFA device by searching for peaks in pixel intensity.
 4. Thesmartphone-based LFA reader of claim 3, wherein said reader determinesthe mean or median grayscale intensity of each line and, optionally, thesum of all test line intensities.
 5. The smartphone-based LFA reader ofclaim 4, wherein said reader compares the measured line intensities withan internal standard curve and displays the concentration of βTP in thesample applied to the sample addition zone, and/or a clinical diagnosis.6. The smartphone-based LFA reader of claim 1, wherein said detectionzone comprises a third test line (T3) that comprises binding moietiesthat bind a complex formed between beta-trace protein (βTP) and anindicator attached to a βTP binding molecule, wherein: said first testline (T1) said second test line (T2) and said third test line (T3) areconfigured so that either no test line signal or just a signal at saidfirst test line (T1) is detectable by said reader when βTP concentrationin a sample applied to said device is lower than or equal to the βTPlevel indicative of the absence of a CSF leak; said first test line (T1)said second test line (T2) and said third test line (T3) are configuredso that a signal is detectable by said reader at said first test lineand said second test line, but not at said third test line when βTPconcentration in a sample applied to said device is greater than the βTPlevel indicative of no CSF leak and less than a βTP level definitive ofthe presence of a CSF leak; and a signal is detectable by said reader atall three test lines T1, T2, and T3 when βTP concentration in a sampleapplied to said device is equal to or greater than a βTP leveldefinitive of the presence of a CSF leak.
 7. The smartphone-based LFAreader of claim 6, wherein said first test line (T1) said second testline (T2) and said third test line (T3) are configured so that no signalis detectable by said reader at test lines T1, T2, or T3, or a signal isdetectable only at test line T1, when βTP concentration is less thanabout 0.7 mg/L in a nasal drip sample applied to said lateral flowdevice.
 8. The smartphone-based LFA reader of claim 6, wherein saidfirst test line (T1) said second test line (T2) and said third test line(T3) are configured so a signal is detectable by said reader at testlines T1, and T2, but not at test line T3, when βTP concentration rangesfrom about 0.7 mg/L up to about 1.3 mg/L in a nasal drip sample appliedto said device.
 9. The smartphone-based LFA reader of claim 6, whereinsaid first test line (T1) said second test line (T2) and said third testline (T3) are configured so a signal is detectable by said reader attest lines T1, T2, and T3, when βTP concentration is greater than about1.3 mg/L in a nasal drip sample applied to said device.
 10. Thesmartphone-based LFA reader of claim 6, wherein said lateral flow devicecomprise a control line downstream from said first test line and saidsecond test line, wherein said control line comprises binding moietiesthat can capture the said indicator, in the presence or absence of βTP.11. The smartphone-based LFA reader of claim 1, wherein said lateralflow device comprises a conjugation zone comprising said indicatorattached to a βTP binding molecule, where said conjugation zone isdisposed in said sample addition zone and/or between said sampleaddition zone and said detection zone.
 12. The smartphone-based LFAreader of claim 1, wherein said lateral flow device comprises anabsorbent pad disposed downstream from said detection zone and when acontrol line is present said absorbent pad is disposed downstream fromsaid control line.
 13. The smartphone-based LFA reader of claim 1,wherein said indicator is selected from the group consisting of acolorimetric indicator, a fluorescent indicator, a radioactiveindicator, and a magnetic indicator.
 14. The smartphone-based LFA readerof claim 1, wherein said βTP binding molecule is an antibody or antibodyfragment.
 15. The smartphone-based LFA reader of claim 1, wherein saidbinding moieties that bind a complex formed between beta-trace protein(βTP) and an indicator comprise an antibody or antibody fragment. 16.The smartphone-based LFA reader of claim 15, wherein said bindingmoieties that bind a complex formed between beta-trace protein (βTP) andan indicator comprise an antibody that specifically binds βTP.
 17. Thesmartphone-based LFA reader of claim 1, wherein said lateral flow deviceis configured to perform a sandwich assay.
 18. The smartphone-based LFAreader of claim 1, wherein, said lateral flow device comprises a poroussubstrate comprising a material selected from the group consisting ofsintered glass or sintered ceramic, a mineral, cellulose, a fiberglass,a nitrocellulose, polyvinylidene fluoride, a nylon, a charge modifiednylon, a polyethersulfone, and combinations thereof.
 19. A kit forsemi-quantification of βTP in a biological sample, said kit comprising:a smartphone-based LFA reader of claim 1; and a container containing alateral flow device for the semi-quantitative detection of acerebrospinal fluid.
 20. The kit of claim 19, wherein said kit containstwo barcode-style LFA test devices, one for testing serum and the otherfor testing a nasal drip sample.